伪狂犬病病毒Ea株EP0基因的克隆序列分析及其在大肠杆菌中的表达  

Cloning,Sequence Analysis and Expression in E.coli of the EP0 Gene of Pseudorabies Virus Ea Strain

作　　者：方六荣[1] 陈焕春[1] 肖少波[1] 马相如[1] 王革飞[1] 

机构地区：[1]华中农业大学牧医学院动物病毒室,湖北武汉430070

出　　处：《中国病毒学》2001年第2期183-187,共5页Virologica Sinica

基　　金：国家自然科学基金!资助项目 (39970 5 5 9)

摘　　要：以伪狂犬病病毒Ea株 (PRV Ea)细胞感染物为模板 ,PCR扩增出 1.2 3kb的EP0基因完整编码区片段 ,将该基因片段克隆到 pBluescriptⅡsk +中并构建三个测序质粒 ,双脱氧末端终止法序列测定并同国外InFh株进行同源比较 ,发现PRV EaEP0基因存在多处点突变和一处缺失突变 ,但无插入突变和移码。进一步将该片段插入到原核表达载体 pET 2 8a的His Tag下游 ,构建的原核表达质粒pETEP0在大肠杆菌BL2 1(DE3)中获得了高效表达 ,SDS PAGE结果显示 ,表达的蛋白分子量为 6 2kD ,并形成包涵体。Western印迹分析表明 ,该蛋白带能与Ea株野毒高免血清发生特异性反应 ,证实PRV EaEP0基因在BL2 1(DE3)中获得了正确表达 ,并且具有免疫学活性。为今后深入研究该基因的功能奠定了基础。The 1.23 kb DNA fragment encoding the early protein EP0 of pseudorabies virus (PRV) Ea strain was amplified by PCR technique and cloned into pBluescriptII sk+.Three sequencing plasmids containing the partial fragment of the EP0 gene were constructed and the sequences were obtained by Sanger's sequencing technique. Compared with PRV InFh strain, there were multipile site mutations and a deleted mutation in the EP0 gene of PRV strain Ea,and the diversity of amino acid residues also existed.Then, the EP0 gene was inserted into an expression vector, pET 28a, fused into the downstream of the 6ΧHis Tag in frame, to yield the expression plasmid pETEP0. After induction by IPTG, a high expression of fusion protein was obtained, SDS PAGE analysis and Western blotting showed that the fusion protein was 62kD and the protein was specific to antisera against PRV Ea strain. This indicated that the EP0 gene be expressed in BL 21 (DE 3) and the expression products have immuno genicity.

关 键 词：伪狂犬病病毒 EP0基因克隆 序列分析 表达 

分 类 号：S855[农业科学—临床兽医学]